A new acrylamido buffer has been synthesized, for use in isoelectric focusing in immobolized pH gradients. This compound (2-acrylamido glycolic acid) has a pK = 3.1 (at 25°C, 20 mM concentration during titration) and is used, by titration with the pK 9.3 Immobiline, to produce a linear pH gradient in the pH 2.5-3.5 interval. Pepsin (from pig stomach) focused in this acid pH gradient is resolved into four components, two major (with pI values 2.76 and 2.78) and two minor (having pI values 2.89 and 2.90). This is the first time that such strongly acidic proteins could be focused in an immobilized pH gradient. Even in conventional isoelectric focusing in amphoteric buffers it has been impossible to focus reproducibly very-low-pI macromolecules.
Focusing of pepsin in strongly acidic immobilized pH gradients / P.G.Righetti, M. Chiari, P.K. Sinha, E. Santaniello. - In: JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS. - ISSN 0165-022X. - 16:2-3(1988), pp. 185-192.
Focusing of pepsin in strongly acidic immobilized pH gradients
E. SantanielloUltimo
1988
Abstract
A new acrylamido buffer has been synthesized, for use in isoelectric focusing in immobolized pH gradients. This compound (2-acrylamido glycolic acid) has a pK = 3.1 (at 25°C, 20 mM concentration during titration) and is used, by titration with the pK 9.3 Immobiline, to produce a linear pH gradient in the pH 2.5-3.5 interval. Pepsin (from pig stomach) focused in this acid pH gradient is resolved into four components, two major (with pI values 2.76 and 2.78) and two minor (having pI values 2.89 and 2.90). This is the first time that such strongly acidic proteins could be focused in an immobilized pH gradient. Even in conventional isoelectric focusing in amphoteric buffers it has been impossible to focus reproducibly very-low-pI macromolecules.
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